The human monoamine transporters (hMATs) for norepinephrine (hNET), dopamine (hDAT), and serotonin (hSERT) are targets for therapeutic drugs (antidepressants) and addictive psycho-stimulants (cocaine, amphetamines). Information about drugs that interact with MATs comes largely from radio-ligand substrate assays. With the Phase I CEBRA application, we will develop a new assay for hMAT drug interactions, in particular the catecholamines, utilizing fluorescent substrates and real-time, single cell fluorescence microscopy and spectroscopy. We plan to analyze hNET and hDAT activity in transfected cells with a novel substrate for the catecholamines, ASP [4-(4-(dimethylamino)styrl)-N-methyl-pyridinium]. ASP is structurally similar to MPP, but ASP fluoresces in the visible range with large Stokes shift (4751 => 610 1). We have already demonstrated that ASP is a Na-dependent substrate with uM affinity for NET and DAT. In Aim I we will measure ASP accumulation in human embryonic kidney cells (HEK) expressing hNET and MAT and measure transporter activity against antidepressants and psycho-stimulants. Anisotropy and quenching of ASP fluorescence can be exploited to distinguish binding from transport and we will test antidepressants, cocaine, and amphetamines against hNET and hDAT to examine which processes is affected. In Aim 1 we will first study NET in a native tissue culture system (superior cervical ganglion neurons) and initiate similar studies with DAT. Primary tissue culture also provides a means of examining the impact of endogenous associated proteins, which may be specific neuronal therapeutic targets, avoiding systemic complications. The overarching goal of Aims I and II is to develop the ASP assay for high-throughput screening of putative drugs that inhibit or stimulate monoamine transport and binding, in particular the catecholamines, norepinephrine and dopamine.